Understanding the diverse chemistry displayed by P450s is a central issue in biomedical science and requires understanding their notorious propensity for specificity and promiscuity. Drug metabolizing P450s display a remarkable promiscuity in substrate recognition, while enzymes involved in steroid biosynthesis are often very specific with respect to substrate binding and catalysis. However, the molecular mechanism by which these different forms control this promiscuity or specificity is not well understood. How does structure encode such diversity? It is known that significant conformational changes occur in many, if not all, P450s during substrate recognition. These changes may also play a role in gating the reactions following O2 activation to produce reactive intermediates. However, we know very little about how substrates are recognized by a particular P450 or how these changes are coupled to function. Does a P450 recognize its substrate by induced fit or by dynamically sampling only a few preferred conformations? Results in the past period have produced significant insights into the conformational changes that occur upon substrate binding to P450, they suggest how these changes may be coupled to function, and they provide a platform for development of novel catalysts with designed specificity. The current aims will use a library of molecular probes for the P450 active site to control the formation and properties of proposed reactive intermediates. These studies will test specific hypotheses, based on our recent progress, about how conformational changes at the active site may help control the O2 activation step. We will develop these approaches to investigate the electrochemical behavior of P450s specifically wired to electrode surfaces. Finally, we will explore the potential for using specific probes for molecular evolution of novel catalysts with designed substrate specificity. These studies will contribute to a better understanding how the protein structure of these important enzymes is coupled to function and substrate specificity. PUBLIC HEALTH RELEVANCE: P450s are a diverse class of enzymes of critical importance to human health because they are responsible for the biosynthesis of important compounds and drug metabolism. Our long term goal is to develop a general method for introducing new activities into these enzymes, as evolved P450 catalysts have the potential for producing novel drugs and antibiotics. In the process, we will gain a deeper understanding of how the substrates are recognized and how the enzymatic reactions are controlled.